Surface plasmon resonance (SPR) is an optical phenomenon which occurs as a result of total internal reflection of light at a metal film--liquid interface. Total internal reflection is observed in situations where light travels through a medium such as glass, and is reflected back through that medium from the interface with a different medium, for example a liquid buffer solution. In order for total internal reflection to occur, the angle of incidence of the light must be greater than a critical angle determined by the refractive indices of the optical media. Although the light is totally reflected, a component of the incident light momentum, termed the evanescent wave, penetrates a distance, on the order of one wavelength, into the medium, e.g. the buffer, on the opposite side of the interface.
If the incident light is monochromatic and polarized, and the interface between the media is coated with a thin film of gold or silver having a thickness which is a fraction of the wavelength of the incident light, the evanescent wave can interact with free oscillating electrons, or plasmons, in the metal film surface. The plasmons will absorb energy from the evanescent wave at a particular angle of incidence, which is dependent upon the refractive index of the liquid medium adjacent to the metal film, i.e. within a distance of about 300 nm. from the metal film. Thus, for a given refractive index in the liquid, the intensity of the reflected light varies according to the angle of incidence of the light, and there is a sharp drop in the intensity of the reflected light at a particular angle at which peak absorbance occurs. This angle can be termed the "resonance angle." Changes in the refractive index of the buffer solution will alter the resonance angle. By measuring the angle at which the peak occurs, it is possible to detect changes in the refractive index of the buffer solution.
Because proteins in the buffer solution alter its refractive index, it is possible to measure, and monitor continuously, the protein content in the buffer solution adjacent to the metal film by measuring the resonance angle. The interaction of macromolecules in the buffer solution with surface immobilized ligand, e.g., antibody binding to peptide or protein, causes a change in the refractive index. This change results in a correlative change in the resonance angle which is detected and quantitated.
Surface plasmon resonance technology is utilize at in commercially available instruments, for example, the BIAcore.RTM. SPR detector manufactured by Pharmacia Biosensor AB (Uppsala, Sweden).
The BIAcore.RTM. apparatus uses surface plasmon resonance to measure the binding affinity and avidity of selected ligates, e.g., immunoglobulins or other proteins and peptides, with an immobilized ligand of interest, e.g. an antibody.
The methodology relies on immobilization of ligands onto the surface of a sensor chip consisting of a glass substrate having a gold film covered by a monolayer of a long hydroxyalkyl thiol to which is covalently attached a thin layer of carboxymethylated dextran. The immobilization procedure is perforated with the sensor chip in place in the instrument and is continuously monitored by the SPR detector. In this procedure, the carboxyl groups of the carboxymethyl dextran are activated by injection of a mixture of N-ethyl-N'-(dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), both prepared in water. In a second step, the ligand (protein) is injected over the surface in a low ionic strength solution at a pH below the isoelectric point of the protein. Since only a fraction of the carboxyl groups are activated by the EDC/NHS mixture, the positively charged protein concentrates onto the surface of the sensor chip via electrostatic attraction, and simultaneously, the amines of the protein react with the NHS-esters resulting in the formation of amide-links between the protein and the dextran surface [S. Loas et al, J. Chem. Soc., Chem. Commune., 1526-1528 (1990)].
Residual NHS-esters remaining after ligand immobilization are then reacted with a solution of ethanolamine. Finally, the surface is subjected to an acid wash to remove non-covalently adsorbed protein. The entire immobilization procedure, which typically takes less than 30 minutes, can be controlled by parameters such as protein concentration, protein solution ionic strength and pH, reagent (EDC/NHS) concentration and reaction times.
The sensor chip is contacted by a microfluidic cartridge which has formed on it a number of channels (typically four) which define the flow of samples across the surface of the sensor chip. The microfluidic cartridge, which is in place when the ligand is introduced to the sensor, contains pneumatic valves which control the flow of samples through the channels.
An unknown sample or ligate solution is introduced into the apparatus to contact the immobilized ligand. The interaction between ligand and ligate is observed directly by surface plasmon resonance techniques and the measurements recorded on a computer via a program such as Bialogue [Pharmacia].
In the conventional surface plasmon resonance instrument, the ligate is discarded to waste after interacting with the ligand.